Leishmaniasis is a major and severe parasitic disease that affects humans, canines (dogs, wolves, foxes, coyotes, jackals), and to a lesser degree, felines (lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx).
Leishmania Leishmania and Leishmania Viannia subgenera are grouped into complexes of species and subspecies based upon molecular, biochemical and immunological similarities. There are several forms of the disease named by their clinical presentation including cutaneous, mucocutaneous or visceral leishmaniasis. Each of these forms of disease is caused by different species of sand flies found in different regions of the world. Cutaneous leishmaniasis of humans is associated with members of L. major, L. tropica and L. aethiopica complexes in the Old World and L. mexicana and L. braziliensis complexes in the New World. Visceral leishmaniasis is caused by L. donovani and L. infantum in Old World regions and by L. infantum in the New World. L. infantum is the primary agent associated with canine leishmaniasis.
The agent of visceral leishmaniasis is a protozoan parasite and belongs to the Leishmania donovani complex. This parasite is widely distributed in temperate and subtropical countries of Southern Europe, Africa, Asia, South America and Central America (Desjeux P. et al.). Leishmania donovani infantum (L. infantum) is responsible for the feline and canine disease in Southern Europe, Africa, and Asia and South and Central America. In humans, the agent is Leishmania donovani donovani (L. donovani), which is also related to L. infantum. 
Sand flies of the genus Phlebotomus (Old World) and Lutzomyia (New World) are the primary vectors responsible for disease transmission. Currently these are the only known vectors capable of spread; fleas, ticks and other arthropods have not been shown to be competent vectors. However, rare cases of leishmaniasis have been contracted through exchange of blood or body fluids, direct contact and at least one case of congenital transmission. P. ariasi, P. perniciosus and P. neglectus are the most common carriers in Southern Europe, Africa, and Asia, whereas Lu. longipalpis is the most common carrier in Southern and Central America.
Canine leishmaniasis is a slowly progressive disease that can take up to 7 years to become clinically apparent (McConkey S E et al.; Slappendel R J et al.). Even then, signs are frequently nonspecific and a diagnosis of Leishmania is seldom considered. Dogs are most commonly infected with L. infantum (L. donovani complex) which is responsible for viscerotropic disease in people. However, up to 90% of infected dogs present with both visceral and cutaneous lesions (Slappendel R J et al.). On the other hand, many dogs appear naturally resistant to this parasite and may remain asymptomatic despite known infection (Grosjean N L et al.). It is estimated that only 10% of dogs residing in endemic areas actually develop clinical disease (Lindsay D S et al.). This lower incidence of clinical disease is attributed to a genetic predisposition of certain dogs to mount a more protective cell-mediated immune response than a humoral response (Lindsay D S et al., McConkey S E et al., Slappendel R J, et al.). Furthermore, it has been reported that up to 20% of infected dogs may mount an adequate immune response and spontaneously recover from clinical illness (McConkey S E et al.). In animals that mount a humoral response, IgG1 appears to correlate with clinical disease while asymptomatic dogs have higher IgG2 antibody levels (Lindsay et al.).
Some of the more frequently reported clinical signs of leishmaniasis include listlessness, fatigue and exercise intolerance coupled with anorexia and weight loss that eventually culminate as wasting disease (McConkey S E et al.). These signs may or may not be accompanied by fever, local or generalized lymphadenopathy (90%) and/or hepatosplenomegaly (Grosjean N L et al., Lindsay D S et al., McConkey S E et al., Martinez-Subiela S et al.). Articular involvement is also fairly common and may present as lameness with swollen joints or simply as a stiff gait. Less common findings include ocular lesions (<5%), chronic diarrhea (30%) and long, deformed brittle nails (20%) referred to as onychogryphosis (Lindsay D S et al., Slappendel R J et al.). Cutaneous lesions are present in up to 89% of infected dogs, with or without overt signs of visceral involvement. Lesions of cutaneous leishmaniasis may occur anywhere on the body but the most common sites are those which are exposed to the environment and are therefore more susceptible to bites from the sand flies. The initial papule rapidly gives rise to an ulcer. Visceral leishmaniasis is invariably fatal if not treated promptly. Visceral leishmaniasis affects the internal body organs, specifically the spleen and the liver.
Dogs are considered the major reservoir of leishmaniasis. The disease is characterized by chronic evolution of viscero-cutaneous signs occurring in less than 50% of infected animals (Lanotte G. et al.). Both asymptomatic and symptomatic dogs with detectable antibodies may be infectious (Molina R. et al.; Courtenay O. et al.). Cats may also be carriers of the protozoan parasites and are thus considered secondary potential reservoirs.
Due to a number of factors, treatment options for leishmaniasis in dogs and response to therapy are limited at best. For some undefined reason, visceral leishmaniasis is more difficult to treat in dogs than in humans. No treatment option is 100% effective in clearing parasitic infection and clinical disease often reappears with cessation of therapy (Lindsay D S et al.). In endemic areas, the most common treatment regimen has been a combination of allopurinol with a pentavalent antimonial such as meglumine antimonite or sodium stibogluconate (Lindsay D S et al., Slappendel R J et al.). However, in recent years this protocol has fallen out of favor due to increasing resistance of the parasite to the drug as well as adverse side effects associated with these compounds (Lindsay D S et al.). To further limit treatment options, Pentostam® (sodium stibogluconate) is the only available antimonial in the United States and its distribution is regulated by the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga. (Lindsay D S et al.).
Mass detection of seropositive dogs followed by culling and/or drug treatment, or the mass application of deltamethrin-impregnated collars, was shown to have an impact in reducing human and canine Leishmaniasis prevalence in endemic areas of Southern Europe, Africa, and Asia (Maroli M. et al. Mazloumi Gavgani A. S. et al.), although the efficacy of eliminating seropositive canines has been debated (Dietze R. et al.; Moreira Jr. E. D. et al.). These control measures are either considered unacceptable, expensive or not effective (Gradoni L. et al.). Mathematical models used to compare the effectiveness of various tools for controlling Leishmaniasis suggest that a canine vaccine may be the most practical and effective method (Dye C.). Therefore, the development of vaccines able to protect canines from Leishmaniasis and/or to prevent disease progression in infected animals is highly desirable for the implementation of Leishmaniasis control programs as well for the veterinary community (Gradoni L. et al.).
However, to date, no vaccine is available for the treatment of Leishmania infection in dogs. The reproducible model of infection of dogs by sand fly bites described in the present invention provides a platform to investigate the efficacy of promising vaccines and will fulfill a long-felt gap in the art. Considering the complexity of canine visceral leishmaniasis, including its chronicity and our lack of understanding of the factors governing virulence and visceralization of Leishmania parasites, the establishment of a protocol for a controlled reproducible visceral infection in dogs that reflects the clinical picture observed in the field represents a valuable tool to undertake vaccine studies, both prophylactic and therapeutic. Importantly, due to the unpredictability of a sand fly transmission season which is influenced by factors such as weather, this transmission model would constitute a significant reduction in cost and a major improvement in study outcomes.
Models for the transmission of parasites by bites of infected sand flies have been established for cutaneous leishmaniasis in mice (Kamhawi et al., 2000; Nathan et al., 2008) and non-human primates (Lawyer et al., 1990; Probst et al., 2001). However, it has been much harder to develop models of transmission by bite for visceral disease. Most models of visceral leishmaniasis rely on the injection of a large number of parasites intravenously or via the intracardiac route (Melby et al, 2001; Wilson et al., 2005). A distinct feature of human leishmaniasis is its polymorphic nature where different parasite species, and for certain species including L. infantum different strains, cause a spectrum of clinical manifestations ranging from asymptomatic, to contained cutaneous infection, to visceral disease (Gradoni & Gramiccia, 1994; First WHO report on neglected tropical diseases, 2010). To date, the factors governing parasite tropism are poorly understood and are thought to be a combination of the genetic susceptibility of the host, virulence of the parasite strain and the immune status of the host. This has likely contributed to the difficulty of developing a model of visceral leishmaniasis by bite of infected sand flies which remains unavailable to date even for small animal models such as mice and hamsters.
More recently, models using intradermal injection with a large number of infectious parasites have been developed (Dondji et al, 2005; Wilson et al., 2005; Gomes et al., 2008). These models however do not resemble the natural mode of infection by sand fly bite where the parasite inoculum is considerably smaller (Kimblin, 2008) and where the transmission event involves the presence of salivary proteins and parasite secretory gel among others, both reported as exacerbating the outcome of disease (Titus & Ribeiro, 1988; Morris et al; 2001; Rogers et al., 2004). Of additional importance, Peters et al. (2009) demonstrated that protection from cutaneous leishmaniasis, observed against needle challenge in vaccinated mice was abrogated by transmission via infected bites. This highlights the importance of using a natural challenge by vector sand fly bites in vaccine-related studies particularly in dogs which are the target animals for canine vaccines.
Taken together, the body of evidence points to a serious and long-felt need for a method that models Leishmania infection, as it occurs naturally in the field (i.e. via the bites of Leishmania-infected sand flies). However, until the instant invention, no one has been able to solve this challenging problem.